Acoustic energy mediation of genetic fragmentation

ABSTRACT

Method and apparatus for controlling acoustic treatment of a sample to mediate a tagmentation process used on double stranded DNA.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.14/881,632, filed Oct. 13, 2015, which claims the benefit of U.S.Provisional Application No. 62/063,683, filed Oct. 14, 2014, whichapplications are hereby incorporated by reference in their entirety.

BACKGROUND 1. Field of the Invention

Systems and methods for processing of samples with acoustic energy aregenerally disclosed.

2. Related Art

Acoustic energy-based sample processing devices, such as AdaptiveFocused Acoustic apparatuses made by Covaris of Woburn, Mass., areeffective for homogenization and disruption of biological tissues, cellsand other sample material. With such devices, a controlled acousticfield enables repeatable processes to be developed which often result inhigher recovery of target molecules. Such target molecules may be, forexample, DNA, RNA, proteins, and the like. Target molecules or othermaterials may be contained as samples within a vessel.

SUMMARY OF INVENTION

Tagmentation is a process in which a hyperactive mutant of the Tn5transposase is used to incorporate a synthetic oligonucleotide intodouble stranded DNA, essentially carrying out a cut/paste procedure inwhich the double stranded DNA is cut, and synthetic sequence isinserted. Its utility in generating libraries for next generationsequencing (NGS) systems was first described in a paper by Andrew Adeyet al. in 2010 (Adey A, Morrison H G, Asan, Xun X, Kitzman J O, Turner EH, Stackhouse B, MacKenzie A P, Caruccio N C, Zhang X, et al. Rapid,low-input, low-bias construction of shotgun fragment libraries byhigh-density in vitro transposition. Genome Biol 11: R119, 2010.) Incommercially available products such as Nextera from Illumina, andMuSeek from Thermo Scientific, the transposase inserts NGSsystem-specific adaptor oligos in the double stranded DNA sample, andsubsequent limited PCR is used to enrich fragments containing thedesired adaptors and barcode indices on either end of the DNA fragments.

The inventors have appreciated that the known tagmentation process hassome well-known limitations, including:

-   -   G+C bias inherent in transposase-mediated fragmentation;    -   Insertion bias towards AT rich region containing similar        insertion nucleic acid sequences described for TN5 transposases;    -   The process is highly sensitive to the DNA input concentration,        requiring precise quantification upstream;    -   DNA fragments distribution after tagmentation is wide, thus        limiting library yield after size selection.

To address at least some of these limitations, the inventors havedeveloped a tagmentation process that is mediated by acoustic energy. Inat least some embodiments, acoustic energy can be used to fragmentdouble stranded DNA having a relatively long base pair length, e.g., inexcess of 3000 bp, e.g., 10000 bp or more. This fragmentation can haveone or more benefits, such as reducing viscosity of the DNA sample whichcan enhance enzymatic activity of the tagmentation process. The acousticenergy can also have other benefits, such as shearing double strandedDNA to a smaller fragment size under 3000 bp, e.g., 1000 bp to 1500 bp.This may help create a narrower DNA fragmentation size after thetagmentation process, thereby enhancing the library yield. In otherembodiments, the acoustic energy treatment may randomly shear segmentsof double stranded DNA material in GC and AT rich regions, and inregions having long repeat portions, so as to form fragments of thedouble stranded DNA material having a base pair length less than 3000bp. This may help reduce the G+C bias inherent in transposase-mediatedfragmentation and/or insertion bias towards AT rich regions containingsimilar insertion nucleic acid sequences described for TN5 transposases.In yet other embodiments, acoustic energy treatment may reduce atransposase concentration required to successfully perform thetagmentation process s, e.g., because the DNA fragmentation caused bythe acoustic treatment and/or mixing caused by the acoustic treatmentmay enhance enzymatic activity of the transposase.

In one aspect of the invention, a method for processing a samplecontaining genomic material includes providing a sample in a vessel, thesample including double stranded DNA material with segments having abase pair length in excess of a starting base pair length. In somecases, the starting base pair length may be greater than 3000 bp ormore, e.g., more than 10000 bp or 48000 bp. The sample may be subjectedto acoustic energy to cause shearing of the segments of double strandedDNA material in the sample to form fragments of the double stranded DNAmaterial having a final base pair length, the starting base pair lengthbeing two or more times the final base pair length. For example, thefragments of DNA material created by acoustic energy treatment may havea base pair length of less than or about 3000 bp, e.g., 1000 to 1500 bp.A hyperactive mutant of Tn5 transposase and oligonucleotide material maybe provided with the fragments of double stranded DNA material to cutthe fragments of double stranded DNA material with the Tn5 transposaseand insert oligonucleotide material into the fragments of doublestranded DNA material at areas cut by the Tn5 transposase. Thetransposase and oligonucleotide material may be provided with the samplebefore or after subjecting the sample to acoustic energy to shear theDNA into fragments. In some embodiments, the sample may be subjected toacoustic energy to mix the hyperactive mutant of Tn5 transposase,oligonucleotide material, and the fragments of double stranded DNAmaterial after the step of subjecting the sample to acoustic energy tocause shearing. This may help enhance the enzymatic and oligonucleotideinsertion process.

In another aspect of the invention, a method for processing a samplecontaining genomic material includes providing a sample in a vessel, thesample including double stranded DNA material with segments having astarting base pair length, and the sample having a starting viscosity.The sample may be subjected to acoustic energy to reduce a viscosity ofthe sample to a reduced viscosity that is less than the startingviscosity. The acoustic energy treatment may also cause shearing of thesegments of double stranded DNA material in the sample to form fragmentsof the double stranded DNA material having a base pair length of lessthan the starting base pair length. A hyperactive mutant of Tn5transposase and oligonucleotide material may be provided with thefragments of double stranded DNA material to cut the fragments of doublestranded DNA material with the Tn5 transposase and insertoligonucleotide material into the fragments of double stranded DNAmaterial at areas cut by the Tn5 transposase. The reduction of viscosityof the sample caused by acoustic energy treatment may enhance theenzymatic activity of the transposase, e.g., because steric hindrancepresence in higher viscosity solutions may be reduced with reducedsample viscosity. As a result, the tagmentation process may be performedmore efficiently. In some embodiments, a concentration of transposaseneeded to perform the tagmentation process may be significantly reducedas compared to tagmentation processes performed without the use ofacoustic energy. The hyperactive mutant of Tn5 transposase andoligonucleotide material may be provided with the fragments of doublestranded DNA material before or after the step of subjecting the sampleto acoustic energy to shear the DNA segments, and in some cases,acoustic energy treatment may be used to mix the hyperactive mutant ofTn5 transposase, oligonucleotide material, and the fragments of doublestranded DNA material after the step of subjecting the sample toacoustic energy to cause shearing. The acoustic energy used to mix maybe provided at a lower power than the acoustic energy used to shear theDNA segments.

In another aspect of the invention, a method for processing a samplecontaining genomic material includes providing a sample in a vessel, thesample including double stranded DNA material with segments having abase pair length in excess of 3000 bp. The segments of double strandedDNA material may be randomly sheared in GC and AT rich regions, and inregions having long repeat portions, to form fragments of the doublestranded DNA material having a base pair length less than 3000 bp. Forexample, in some embodiments, acoustic energy may be employed torandomly shear the DNA segments. A hyperactive mutant of Tn5 transposaseand oligonucleotide material may be provided with the fragments ofdouble stranded DNA material to cut the fragments of double stranded DNAmaterial with the Tn5 transposase and insert oligonucleotide materialinto the fragments of double stranded DNA material at areas cut by theTn5 transposase.

Generally speaking, shearing of DNA and other genomic fragments usingacoustic treatment is known from U.S. Patent Publication 2009/0317884.For example, U.S. Patent Publication 2009/0317884 discloses placing DNAfragments having a base pair length of 10 kbp and up into a 50-100microliter vessel along with an energy director in the form of a polymerrod or bead, and acoustically treating the DNA fragments so as to shearthe DNA fragments into smaller fragment sizes of about 3 kbp. Otheracoustic energy treatment protocols have been provided by Covaris, Inc.of Woburn, Ma. For example, DNA shearing to base pair sizes betweenabout 150 to 1500 bp may be performed using a Covaris S220 system byemploying a peak incident power (PIP) between 140 and 175 Watts, a 2 to10% duty factor, 200 cycles per burst and a treatment time of 15 to 430seconds. Sample volume may be 50 or 130 microliters and may be held in aCovaris microTUBE. (The energy applied to a sample via acoustic energyis measured in Joules and given by the product of peak incident power(PIP in watts) by the duty cycle of the applied energy (DC in percentageterms) by the total processing time (T in seconds) or E=PIP*DC*T).) Aswill be understood, the acoustic energy employed in such shearingoperations is focused acoustic energy such that a focal zone is presentat the sample being treated.

Other advantages and novel features of the invention will becomeapparent from the following detailed description of various non-limitingembodiments when considered in conjunction with the accompanying figuresand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention are described with reference to the followingdrawings in which numerals reference like elements, and wherein:

FIG. 1 shows a schematic block diagram of an acoustic treatment systemthat incorporates one or more aspects of the invention; and

FIG. 2 shows a cross sectional view of a vessel containing a sample thatmay be used with aspects of the invention;

FIG. 3 illustrates schematic steps of a method for performingtagmentation in an illustrative embodiment; and

FIG. 4 shows a vessel used to hold a sample in Example One below.

DETAILED DESCRIPTION

Aspects of the invention are not limited in application to the detailsof construction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. Other embodimentsmay be employed and aspects of the inventions may be practiced or becarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting.

Acoustic treatment systems can be useful for the homogenization anddisruption of biological tissues, cells and other sample material, withthe end goal of recovering target molecules from the sample material,such as DNA, RNA, proteins, and the like. In addition, such systems maybe used along with aspects of the invention for DNA and/or other genomicfragment shearing, e.g., to reduce the base pair length of DNA fragmentsfrom 1,000s or 10,000s of base pairs to lengths of 200-1000 base pairs.As described in more detail below, acoustic energy, and specificallyfocused acoustics, can be useful in mediating a tagmentation process.Generally speaking the inventors have found the following features andbenefits to employing focused acoustics with a tagmentation process:

-   -   Utilizing acoustic energy to randomly shear DNA to an average        size of around 1-1.5 kb may:        -   a. Increase the complexity of the resulting library by            randomly shearing the genomic DNA in GC and AT rich regions,            as well as long stretches of repeats, leading to reduced G+C            bias of the library.        -   b. The 1-1.5 kb fragments will render the fragments more            accessible to the transposases since long stretches of low            concentration genomic DNA are converted into higher            concentration, partially fragmented DNA. The resultant            transposase-processed DNA should have a tighter size            distribution as compared to genomic DNA processed by a            transposase alone.    -   Carrying out the tagmentation reaction in the presence of        acoustic energy, e.g., of relatively low power as compared to        acoustic energy used for shearing, will provide efficient gentle        mixing of the reaction components. As a result, the tagmentation        reaction may experience:        -   a. An increase in the rate of sample/enzyme interaction            leading to a more efficient tagmentation reaction. This can            reduce the concentration of transposase required per            reaction further reducing the cost of library preparation.        -   b. Further reduction in the distribution of DNA fragments            generated by tagmentation as a result of efficient mixing of            the components during the tagmentation reaction.        -   c. Better control over the tagmentation fragment size. This            will allow for greater utility of tagmentation in            applications requiring longer fragment inserts.        -   d. Reduce tagmentation sensitivity to input DNA mass and            concentration.

FIG. 1 shows a schematic block diagram of an acoustic treatment system100 that incorporates one or more aspects of the invention and/or can beemployed with one or more aspects of the invention. It should beunderstood that although embodiments described herein may include mostor all aspects of the invention, aspects of the invention may be usedalone or in any suitable combination with other aspects of theinvention. In this illustrative embodiment, the acoustic treatmentsystem 100 includes an acoustic transducer 14 (e.g., including one ormore piezoelectric elements) that is capable of generating an acousticfield (e.g., at a focal zone 17) suitable to cause mixing, e.g., causedby cavitation, and/or other affects in a sample 1 contained in a vessel4. The acoustic transducer 14 may produce acoustic energy within afrequency range of between about 100 kilohertz and about 100 megahertzsuch that the focal zone 17 has a width of about 2 centimeters or less.The focal zone 17 of the acoustic energy may be any suitable shape, suchas spherical, ellipsoidal, rod-shaped, or column-shaped, for example,and be positioned at the sample 1. The focal zone 17 may be larger thanthe sample volume, or may be smaller than the sample volume, as shown inFIG. 1. U.S. Pat. Nos. 6,948,843 and 6,719,449 are incorporated byreference herein for details regarding the construction and operation ofan acoustic transducer and its control.

The vessel 4 may have any suitable size or other arrangement, e.g., maybe a glass tube, a plastic container, a well in a microtiter plate, avial, or other, and may be supported at a location by a vessel holder12. Although a vessel holder 12 is not necessarily required, the vesselholder 12 may serve to interface with the acoustic processing device sothat the vessel 4 and the sample in the vessel is positioned in a knownlocation relative to an acoustic field, for example, at least partiallywithin a focal zone of acoustic energy. In this embodiment, the vessel 4is a 130 microliter borosilicate glass tube, but it should be understoodthat the vessel 4 may have other suitable shapes, sizes, materials, orother feature, as discussed more below. For example, the vessel 4 may bea cylindrical tube with a flat bottom and a threaded top end to receivea cap, may include a cylindrical collar with a depending flexiblebag-like portion to hold a sample, may be a single well in a multiwellplate, may be a cube-shaped vessel, or may be of any other suitablearrangement. The vessel 4 may be formed of glass, plastic, metal,composites, and/or any suitable combinations of materials, and formed byany suitable process, such as molding, machining, stamping, and/or acombination of processes.

The acoustic treatment system 100 may also include a coupling mediumcontainer 15 that is capable of holding a medium 16 (such as water orother liquid, gas, gel, solid, semi-solid, and/or a combination of suchcomponents) which transmits acoustic energy from the transducer 14 tothe vessel 4. In some embodiments, the acoustic field may be controlled,the acoustic transducer 14 may be moved, and/or the vessel 4 may bemoved (e.g., by way of moving a holder 12, such as a rack, tray,platform, etc., that supports the vessel 4) so that the sample ispositioned in a desired location relative to the focal zone 17. Also,the holder 12 is not limited to a device like that shown in FIG. 1, andinstead may include a rack, slot, tray, gripper element, clamp, box orany other suitable arrangement for holding the vessel, or multiplevessels, in a desired location. For example, the holder 12 may includeone or more multi-vessel supports and a rack. Each support may hold aplurality of vessels 4, e.g., a plurality of vessels may be held in alinear array. Each support may include an identifier, such as a barcode,RFID chip, or other component that may be read so as to identify thesupport and/or vessels 4 associated with the support. The rack may holdmultiple supports with vessels and make it easier to physicallymanipulate or otherwise handle multiple vessels 4, e.g., in an automatedprocessing environment in which one or more robotic devices manipulatevessels for acoustic or other processing. For example, the support mayinclude a strip of material with holes into which each vessel isinserted. The rack may be arranged in the form of a multiwell plate suchthat vessel bottoms extending below the support may be received into acorresponding opening or well of the plate. The rack may also include anidentifier so that the rack and/or supports on the rack can beidentified in a automated way, e.g., by a laser scanner, optical camera,RFID tag reader or other arrangement.

To control the acoustic transducer 14, the acoustic treatment system 100may include a system control circuit 10 that controls various functionsof the system 100 including operation of the acoustic transducer 14. Forexample, the system control circuit 10 may provide control signals to aload current control circuit, which controls a load current in a windingof a transformer. Based on the load current, the transformer may outputa drive signal to a matching network, which is coupled to the acoustictransducer 14 and provides suitable signals for the transducer 14 toproduce desired acoustic energy. As discussed in more detail below, thesystem control circuit 10 may control various other acoustic treatmentsystem 100 functions, such as positioning of the vessel 4 and/oracoustic transducer 14, receiving operator input (such as commands forsystem operation), outputting information (e.g., to a visible displayscreen, indicator lights, sample treatment status information inelectronic data form, and so on), and others.

In this illustrative embodiment, the sample 1 includes DNA segments 2and a liquid 3, e.g., 15 to 130 microliters of liquid containing 20-30nanograms of DNA fragments per microliter. (Although the DNA segments 2are shown schematically as a single block of material, this is forpurposes of illustration only. The DNA segments 2 may be dispersed inthe liquid 3 and generally will not form a solid mass.) The DNA segments2 may have a starting base pair length of 3 kbp, 5 kbp, 10 bkp, 48 kbpor more, e.g., such that 90% or more of the DNA fragments have a basepair length over 3 kbp, 5 kbp, etc. Of course, those of skill in the artwill appreciate that the sample 1 is not limited to including a liquid3, as the sample 1 may take any suitable form, such as a solid onlyform, a gel, a semi-solid, etc.

In at least some embodiments, the sample volume may be less than thevolume of the vessel, and thus an interface 5 will separate the sample 1from a headspace 6 in the vessel, i.e., a gaseous region immediatelyabove the sample 1. This arrangement may cause portions of the sample 1to be splashed or otherwise ejected from the interface 5 in someconditions, e.g., to adhere to the vessel 4 sidewalls above theinterface 5. However, the presence of one or more beads 8 in the sample1 may reduce splashing or other sample 1 ejection from the interface 5.The beads 8 may function as a nucleation site for cavitation induced bythe acoustic energy and cause shear forces created during cavitationbubble collapse to be directed to the surface of the bead, instead of toother portions in the sample. By arranging all portions of the samplewithin a maximum distance, e.g., 2 mm or less, of a bead surface, allportions of the sample may be positioned suitably near the bead surface(e.g., due to mixing during acoustic treatment) to experience shear orother forces that cause shearing of genomic material.

Thus, in some embodiments, the presence of the bead(s) 8 in the sample 1having a volume of 1-30 microliters may enable shearing of DNA to occurunder lower power or energy conditions than would otherwise be possible.In addition, DNA or other genomic segments may be sheared to lengthsmuch shorter than previously possible under relatively low power orenergy conditions. DNA segments having a base pair length of 3 kbp, 5kbp, 10 bkp, 48 kbp or more may be sheared by acoustic energy having aPIP of 20 watts or less and a duty cycle of 10-20% in 30-200 secondssuch that the 90%, 95% or more of the fragments end up with a base pairlength of 200-1500 bp. This is a significant and surprising improvementover prior processes.

The bead(s) may have a diameter of about 1-3 mm, with a diameter of 1.57mm being found particularly effective in genomic shearing with samplevolumes around 15 microliters. In some embodiments, three beads 8 havinga 1.57 mm diameter have been found particularly useful. Beads made ofPTFE have been found effective, although other polymer materials areexpected to work as well. Beads having a coarse surface finish, asopposed to a polished surface finish, have been found to be effective inmany applications. Generally, the beads are non-buoyant so as to remainimmersed in the sample during acoustic processing, but the beads couldbe formed as part of a vessel wall, be attached to a vessel wall, or beneutrally buoyant. Also, although beads 8 in the illustrated embodimentare shown as spherical in shape, the beads may have a variety of shapes,e.g., like jewelry elements commonly referred to as “beads” have avariety of different shapes and sizes. However, it should be understoodthat the use of beads 8 is not required, and instead, DNA shearing maybe performed in the absence of any beads 8 or other elements in thevessel 4.

The embodiment shown in FIG. 1 also includes a cap 9 that may be used toclose the open end of the vessel 4. By capping the open end of thevessel 4, an operator may be able to prevent flow out of/into the vessel4 and/or prevent contamination of the sample 1 by the outsideenvironment. The cap 9 may engage the vessel 4 in any way, such as byscrew thread, interference fit, frictional engagement to the inner orouter surface of the vessel sidewall, etc. The use of a cap 9 isoptional, and not required.

In one aspect of the invention, a vessel in which genomic material issheared may have a conically shaped bottom arranged such that theconical walls diverge upwardly from each other at an aperture angle ofabout 12-20 degrees. For example, FIG. 2 shows an arrangement in whichthe sidewalls 41 of the vessel 4 diverge from each other at an apertureangle α of about 16 degrees. The sidewalls 41 may be relatively thin,e.g., about 0.25 mm in thickness, so as to reduce interference withacoustic energy and/or enhance heat exchange with the coupling medium15. The extreme bottom 42 of the vessel may have a partial sphericalshape with a radius of about 1-2 mm. The bottom 42 may be thicker thanthe sidewalls 41 as shown, or may have the same or smaller thickness.The partial spherical bottom may help with recovery of sample afterprocessing, since sample may tend to collect at the bottom, allowingpipetting from the vessel. The sample 1 may have a height h in thevessel 4 of about 3-4 mm as measured from the inner bottom of the vesselto the interface 5.

FIG. 3 outlines steps in a method of performing a tagmentation processwith genomic material in accordance with aspects of the invention. Avessel 4 is provided having a vessel volume, which may be 50-150microliters or more (or less). The vessel 4 may be made of glass or apolymer or other material, if suitable. Glass materials may aid in heattransfer to a coupling medium, and some polymer materials have beenfound to aid in genomic material shearing. The vessel 4 may have aconically-shaped bottom with sidewalls upwardly diverging at an angle ofabout 12-20 degrees, e.g., as shown in FIG. 2. The extreme bottom of thevessel 4 may have a partial spherical shape. This arrangement has beenfound particularly useful in shearing DNA in a sample volume of about 15microliters. Alternately, the vessel 4 may be a cylindrical tube withvertical sidewalls and a flat or spherical bottom, as shown.

Next, a sample 1 containing DNA segments 2 and liquid 3 may be placed inthe vessel 4. The sample 1 may have a volume of about 1 to 130microliters, with a volume of 15 microliters having been found in someexamples to be particularly suitable for effective DNA shearing. Thesample 1 may have a height in the vessel 4 of about 3-4 mm above thevessel inner bottom. Genomic fragments in the sample may be provided ata concentration of about 20-30 nanograms/microliter, and may havefragment lengths of more than 3 kbp, 5 kbp, 10 kbp, 48 kbp or more.Optionally, one to three polymer beads, e.g., made of PTFE having adiameter of 1-3 mm may be provided in the sample 1 so the beads areimmersed in the sample. Providing three spherical PTFE beads having adiameter of about 1.57 mm in a 15 microliter sample has been foundparticularly suitable for shearing DNA.

Thereafter, the sample 1 may be treated with acoustic energy to shearthe DNA segments in the sample 1. The acoustic energy may have afrequency range of between about 100 kilohertz and about 100 megahertzand have a focal zone 17 with a width of about 2 centimeters or less.The focal zone 17 may be positioned so that the entire sample 1 islocated in the focal zone 17, or so that a portion of the sample is inthe focal zone 17. In some embodiments, the sample may move through thefocal zone 17, whether by moving the focal zone 17 relative to thevessel 4 or moving the vessel 4 relative to the focal zone 17. Theacoustic energy may have a peak incident power (PIP) of 20 watts or lessand a duty cycle of 10-20%. The sample 1 may be treated with theacoustic energy over a time period of 30-200 seconds. As a result, 90%,95%, 99% or more of the genomic material having a relatively longstarting base pair length may be sheared to fragments having a resultingbase pair length that is at least one-half or smaller than the startingbase pair length, e.g., 1000 to 1500 bp. In some embodiments, 99% ormore of the initial genomic material may be sheared to have a base pairlength of 1000-1500 bp.

With acoustic energy shearing complete, a hyperactive mutant of Tn5transposase and oligonucleotide material may be provided with thefragments of double stranded DNA material to cut the fragments of doublestranded DNA material with the Tn5 transposase and insertoligonucleotide material into the fragments of double stranded DNAmaterial at areas cut by the Tn5 transposase. The transposase andoligonucleotide material may be provided with the sample by pipette 11or in other ways, and may be provided before or after subjecting thesample to acoustic energy to shear the DNA into fragments. That is, theDNA segments may be sheared by acoustic energy while in the presence ofthe transposase and olionucleotide material. In some embodiments, thesample may be subjected to acoustic energy to mix the hyperactive mutantof Tn5 transposase, oligonucleotide material, and the fragments ofdouble stranded DNA material after the sample is exposed to acousticenergy to cause shearing. This may help enhance the enzymatic andoligonucleotide insertion process. In other embodiments, once acousticenergy shearing and tagmentation is complete, other processes may beperformed on the sample while in the vessel, such as PCR amplification,stirring, catalyzing, heating, disruption of molecular bonds, or anyother appropriate process. Such processes may be performed usingacoustic energy, or not, e.g., PCR processing may be performed by astandard thermocycler machine.

A few illustrative examples of DNA shearing and tagmentation processesusing methods and systems according to the invention are describedbelow.

Example One

A vessel having a volume of 130 microliters was provided with a 15microliter sample containing lambda DNA (i.e., DNA fragments having abase pair length of 48 kbp or more) at a concentration of about 28nanograms/microliter, but may be lower, e.g., down to 7nanograms/microliter. Three 1.57 mm PTFE beads were provided in thesample as well, and the borosilicate glass vessel having a sphericalbottom was closed by a split septum, as shown in FIG. 4. (Dimensions inFIG. 4 are in millimeters.) The sample was acoustically treated using aCovaris S220 ultrasonicator set to provide a PIP of 18 watts, a 20% dutycycle and 50 cycles per burst for 60 seconds. Some splashing of thesample was observed during acoustic treatment. More than 95% of thelambda DNA fragments were sheared to DNA fragments having a length under1000 bp, with an average base pair length of the sheared DNA being about336 bp. More than 75% of the sheared DNA had a base pair length of100-500 bp. Also of note is that more than 96% of the sample wasrecovered from the vessel after acoustic treatment, and more than 93% ofthe DNA material initially provided in the vessel was recovered bypipetting. The experiment was repeated 12 times, and a coefficient ofvariation of less than 4% was determined, i.e., the results of DNAshearing were found to be highly repeatable and consistent. The totalenergy of about 216 Joules to shear the lambda DNA is far less thanexpected, and is thought to be due at least in part to the relativelysmall sample size and the presence of three beads in the sample.

Example Two

Example One was repeated, except that the samples were processed withtwo 1.57 mm beads and with one 1.57 mm beads. (Processing time for thesingle bead experiment was increased from 60 seconds to 120 seconds.) Inboth experiments, more than 90% of the lambda DNA fragments were shearedto DNA fragments having a length under 1000 bp, with an average basepair length of the sheared DNA being about 332 bp for two beads, andabout 412 bp for one bead. The coefficient of variation for two beadswas about 4.0%, and for one bead was about 7.2% based on 12 repeatexperiments for each.

Example Three

Example One was repeated, except that the samples were processed usingone 2.36 mm bead, i.e., a larger bead than those used in Examples Oneand Two. The results are shown in FIG. 7. More than 95% of the lambdaDNA fragments were sheared to DNA fragments having a length under 1000bp, with an average base pair length of the sheared DNA being about 337bp. The coefficient of variation was about 10.5% based on 12 repeatexperiments, i.e., a significant drop in repeatability versus thesmaller bead sizes. Recovery of the sample volume was about 80%, alsosignificantly less than that found in the three bead example above.

Example Four

Double stranded DNA having segments with a starting base pair size ofabout 5000 bp is sheared using a Covaris S220 system and employing aCovaris protocol to shear DNA to a resulting base pair size of about1500 bp. For example, 2 micrograms of E. coli double stranded DNA isprovided in a 130 microliter sample including water in a CovarismicroTUBE having a 130 microliter volume. A Tris EDTA buffer having a pHof 8.0 is included along with a Covaris microTUBE fiber. The sample istreated with focused acoustic energy from the S220 system employing apeak incident power (PIP) of 140 Watts, a 2% duty factor, 200 cycles perburst and a treatment time of 15 seconds. This treatment shears thedouble stranded DNA to create fragments having a resulting base pairsize of about 1500 bp. Prior to acoustic energy treatment, Dnase Ienzyme is used to nick the input DNA sample material using an enzymeconcentration of 0.1 to 0.001 units and an incubation time of 5 to 60minutes. The Dnase I enzyme activity is stopped by adding EDTA in afinal concentration of 10 mM to the sample and heating to 70 degrees C.for 15 minutes. After acoustic treatment to shear the DNA, the NexteraXT protocol is followed in which a hyperactive mutant of Tn5 transposaseand oligonucleotide material is provided with the fragments of doublestranded DNA material to cut the fragments of double stranded DNAmaterial with the Tn5 transposase and insert oligonucleotide materialinto the fragments of double stranded DNA material at areas cut by theTn5 transposase. An Agilent Bioanalyzer and associated high sensitivityDNA assay is used to analyze aliquots of sample material for fragmentsize distribution. A Kapa library quantification kit is used to measurelibrary efficiency.

As described above, the system control circuit 10 may include anysuitable components to perform desired control, communication and/orother functions. For example, the system control circuit 10 may includeone or more general purpose computers, a network of computers, one ormore microprocessors, etc. for performing data processing functions, oneor more memories for storing data and/or operating instructions (e.g.,including volatile and/or non-volatile memories such as optical disksand disk drives, semiconductor memory, magnetic tape or disk memories,and so on), communication buses or other communication devices for wiredor wireless communication (e.g., including various wires, switches,connectors, Ethernet communication devices, WLAN communication devices,and so on), software or other computer-executable instructions (e.g.,including instructions for carrying out functions related to controllingthe load current control circuit as described above and othercomponents), a power supply or other power source (such as a plug formating with an electrical outlet, batteries, transformers, etc.), relaysand/or other switching devices, mechanical linkages, one or more sensorsor data input devices (such as a sensor to detect a temperature and/orpresence of the medium 16, a video camera or other imaging device tocapture and analyze image information regarding the vessel 4 or othercomponents, position sensors to indicate positions of the acoustictransducer 14 and/or the vessel 4, and so on), user data input devices(such as buttons, dials, knobs, a keyboard, a touch screen or other),information display devices (such as an LCD display, indicator lights, aprinter, etc.), and/or other components for providing desiredinput/output and control functions.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.

The use of “including,” “comprising,” “having,” “containing,”“involving,” and/or variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

While aspects of the invention have been described with reference tovarious illustrative embodiments, such aspects are not limited to theembodiments described. Thus, it is evident that many alternatives,modifications, and variations of the embodiments described will beapparent to those skilled in the art. Accordingly, embodiments as setforth herein are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit of aspects of theinvention.

What is claimed is:
 1. A method for processing a sample containinggenomic material, comprising: providing a sample in a vessel, the sampleincluding double stranded DNA material with segments having a startingbase pair length, the sample having a starting viscosity; subjecting thesample to acoustic energy to reduce a viscosity of the sample to areduced viscosity that is less than the starting viscosity and to causerandom shearing of the segments of double stranded DNA material in thesample to form fragments of the double stranded DNA material having abase pair length of less than the starting base pair length; andproviding a hyperactive mutant of Tn5 transposase and oligonucleotidematerial with the fragments of double stranded DNA material to cut thefragments of double stranded DNA material with the Tn5 transposase andinsert oligonucleotide material into the fragments of double strandedDNA material at areas cut by the Tn5 transposase.
 2. The method of claim1, wherein the step of providing a hyperactive mutant of Tn5 transposaseand oligonucleotide material with the fragments of double stranded DNAmaterial occurs before the step of subjecting the sample to acousticenergy.
 3. The method of claim 1, further comprising: subjecting thesample to acoustic energy to mix the hyperactive mutant of Tn5transposase, oligonucleotide material, and the fragments of doublestranded DNA material after the step of subjecting the sample toacoustic energy to cause shearing.
 4. The method of claim 1, wherein thestep of providing a hyperactive mutant of Tn5 transposase andoligonucleotide material with the fragments of double stranded DNAmaterial occurs after the step of subjecting the sample to acousticenergy.
 5. The method of claim 4, further comprising: subjecting thesample to acoustic energy to mix the hyperactive mutant of Tn5transposase, oligonucleotide material, and the fragments of doublestranded DNA material after the step of subjecting the sample toacoustic energy to cause shearing.
 6. The method of claim 1, wherein theoligonucleotide material includes synthetic oligonucleotides.
 7. Themethod of claim 1, wherein the sample has a volume of about 15microliters.
 8. The method of claim 1, wherein the vessel has a volumeof about 100 microliters.
 9. The method of claim 1, wherein the startingbase pair length is in excess of 10000 bp.
 10. The method of claim 1,wherein the fragments of the double stranded DNA material have a basepair length less than 3000 bp.
 11. The method of claim 1, wherein thefragments of the double stranded DNA material have a base pair lengthbetween 1000 bp and 1500 bp.
 12. The method of claim 1, wherein the stepof subjecting the sample to acoustic energy reduces a viscosity of thesample so as to enhance enzyme interaction that occurs during the stepof providing a hyperactive mutant of Tn5 transposase and oligonucleotidematerial.
 13. The method of claim 1, wherein the subjecting step isperformed over a time period of 30-200 seconds.
 14. The method of claim1, wherein the step of providing a hyperactive mutant of Tn5 transposaseand oligonucleotide material with the fragments of double stranded DNAmaterial occurs after the step of subjecting the sample to acousticenergy.
 15. The method of claim 14, further comprising: subjecting thesample to acoustic energy to mix the hyperactive mutant of Tn5transposase, oligonucleotide material, and the fragments of doublestranded DNA material after the step of subjecting the sample toacoustic energy to cause shearing.